In vivo method for generating diversity in a protein scaffold

Abstract

A transgenic non-human animal is provided. In certain embodiments, the animal comprises a genome comprising an immunoglobulin heavy chain locus comprising: a) a transcribed gene encoding a fusion protein comprising, from N-terminus to C-terminus: i. a scaffold comprising a first binding domain; and ii. a heavy chain constant region operably linked to the scaffold; wherein the scaffold is capable of specifically binding to a target in the absence of additional polypeptides; and b) a plurality of pseudogenes that are operably linked to the transcribed gene and that donate, by gene conversion, nucleotide sequence to the part of the transcribed gene that encodes the binding domain.

What is claimed is: 
     
       1. A transgenic chicken comprising a genome comprising an immunoglobulin heavy chain locus comprising:
 a) a transcribed gene encoding a fusion protein comprising, from N-terminus to C-terminus:
 i. a scaffold comprising a first binding domain; and operably linked to said scaffold: ii. a heavy chain constant region that is native to said animal; 
 wherein said scaffold is capable of specifically binding to a target in the absence of additional polypeptides; and 
 
 b) a plurality of pseudogenes that are operably linked to said transcribed gene and that donate, by gene conversion, nucleotide sequence to the part of said transcribed gene that encodes said binding domain, wherein the pseudogenes are upstream or downstream of the transcribed gene and contain a nucleotide sequence that is at least 80% identical to at least part of the transcribed gene. 
 
     
     
       2. The transgenic chicken of  claim 1 , comprising an immunoglobulin light chain locus that encodes a light chain constant region but not a light chain variable domain. 
     
     
       3. The transgenic chicken of  claim 1 , comprising an immunoglobulin light chain locus comprising:
 a) a second transcribed gene encoding a second fusion protein comprising, from N-terminus to C-terminus:
 i. a second scaffold comprising a second binding domain; and 
 ii. a light chain constant region operably linked to said scaffold; 
 wherein said scaffold is capable of specifically binding to a target in the absence of additional polypeptides; and 
 
 b) a plurality of pseudogenes that are operably linked to said second transcribed gene and that donate, by gene conversion, nucleotide sequence to the part of said second transcribed gene that encodes said second binding domain, wherein the pseudogenes linked to the second transcribed gene are upstream or downstream of the second transcribed gene and contain a nucleotide sequence that is at least 80% identical to at least part of the second transcribed gene. 
 
     
     
       4. The transgenic chicken of  claim 3 , wherein said first and second binding domains have different binding specificities. 
     
     
       5. The transgenic chicken of  claim 1 , wherein said scaffold is single-chain antibody. 
     
     
       6. The transgenic chicken of  claim 5 , wherein said single-chain antibody comprises heavy and light chains from a classical antibody that are linked in a single polypeptide chain. 
     
     
       7. The transgenic chicken of  claim 5 , wherein said single-chain antibody is a VHH (variable heavy-heavy) antibody. 
     
     
       8. The transgenic chicken of  claim 7 , wherein said single-chain antibody is a humanized VHH antibody. 
     
     
       9. The transgenic chicken of  claim 1 , wherein said scaffold is not antibody derived. 
     
     
       10. The transgenic chicken of  claim 5 , wherein the nucleotide sequences of said pseudogenes are at least 80% identical to the nucleotide sequence of said part of said transcribed gene. 
     
     
       11. The transgenic chicken of  claim 1 , wherein said immunoglobulin heavy chain locus comprises at least 10 of said pseudogenes. 
     
     
       12. The transgenic chicken of  claim 1 , wherein said transgenic animal is made by replacing the endogenous variable region in an endogenous immunoglobulin heavy chain locus of the transgenic animal with a nucleic acid construct comprising said plurality of pseudogenes and encoding said scaffold, without replacing the constant region of said endogenous immunoglobulin heavy chain locus. 
     
     
       13. The transgenic chicken of  claim 1 , wherein at least one of said plurality of pseudogenes is in reverse orientation relative to said transcribed gene. 
     
     
       14. The transgenic chicken of  claim 1 , wherein said animal is a chicken, rabbit, cow, sheep or goat. 
     
     
       15. A method comprising:
 immunizing a transgenic chicken of  claim 1  with an antigen; and 
 obtaining from said transgenic animal a fusion protein that is encoded by said immunoglobulin heavy chain locus and that specifically binds to said antigen. 
 
     
     
       16. The method of  claim 15 , further comprising:
 making hybridomas using cells of said transgenic chicken; and 
 screening said hybridomas to identify a hybridoma that produces an scaffold protein that specifically binds to said antigen. 
 
     
     
       17. The method of  claim 15 , further comprising using PCR to amplify nucleic acid that encodes said scaffold from a lymphocyte of said transgenic chicken, and expressing a recombinant scaffold protein using said amplified nucleic acid. 
     
     
       18. The method of  claim 17 , further comprising:
 humanizing said scaffold protein.